Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response.

The cell adhesion molecule CD44 has been implicated in diverse biological functions including the pathological responses to infections and inflammatory diseases. The variable forms of CD44 contribute to functional variations, which are not yet defined in teleost. Here, we show that zebrafish CD44a plays a protective role in the host defense against Edwardsiella piscicida infection. Zebrafish CD44a deficiency inhibits cell growth and proliferation, impairs cell growth and death pathways, and regulates the expression levels of many genes involved in p53 signaling, apoptosis and autophagy. In addition, CD44a gene disruption in zebrafish leads to inhibition of apoptosis and induction of autophagy, with the increased susceptibility to E. piscicida infection. Furthermore, we show that zebrafish CD44a variants including CD44a_tv1 and CD44a_tv2 promote the translocation of p53 from the nucleus to the cytoplasm and interact with p53 in the cytoplasm. Mechanistically, zebrafish CD44a_tv1 mediates the beneficial effect for larvae survival infected with E. piscicida is depending on the CASP8-mediated apoptosis. However, the antibacterial effect of zebrafish CD44a_tv2 depends on the cytoplasmic p53-mediated inhibition of autophagy. Collectively, our results identify that different mechanisms regulate CD44a variants-mediated antibacterial responses.

An ultra-sensitive "turn-off" fluorescent sensor for the trace detection of rifampicin based on glutathione-stabilized copper nanoclusters.

Rifampicin is a common antibiotic used in human and veterinary medicine to treat tuberculosis and other diseases caused by numerous pathogenic bacteria. However, the excessive or improper use of rifampicin usually leads to a series of problems, including bacterial resistance, excessive drug-resistance and water pollution. Thus, it is of great importance to develop selective and sensitive assays for monitoring rifampicin in biological systems. In this study, we designed a fluorescence "turn-off" strategy for the trace detection of rifampicin based on a glutathione-stabilized copper nanoclusters (GSH-Cu NC) sensor. In an aqueous solution, the fluorescence of the GSH-Cu NCs at 632 nm can be quenched effectively and selectively by rifampicin due to the inner-filter effect (IFE) of fluorescence mechanism. Distinctively, this GSH-Cu NC sensor exhibited excellent fluorescence sensing capability for the trace detection of rifampicin with a very low limit of detection (LOD) of 16 pM in a wide linear range from 50 to 10 000 pM. It is not only more sensitive than the other methods previously reported for the detection of rifampicin, but also has an outstanding selectivity and strong anti-interference in complex samples. Furthermore, the as-developed GSH-Cu NCs were also successfully applied to determine rifampicin in different real samples with quantitative spike recoveries ranging from 97% to 105%.

Transcriptomic characterization of adult zebrafish infected with Streptococcus agalactiae.

Streptococcus agalactiae is a major aquaculture pathogen infecting various saltwater and freshwater fish. To better understand the mechanism of the immune responses to S. agalactiae in wildtype zebrafish, the transcriptomic profiles of two organs containing mucosal-associated lymphoid tissues from S. agalactiae-infected and non-infected groups were obtained using RNA-seq techniques. In the intestines, 6735 and 12908 differently expressed genes (DEGs) were identified at 24 hpi and 48 hpi, respectively. Among 66 and 116 significantly enriched pathways, 15 and 21 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. A number of genes involved in Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, Antigen processing and presentation, NF-kappa B signaling pathway and PI3K-Akt signaling pathway were significantly downregulated. In the skins, 3113 and 4467 DEGs were identified at 24 hpi and 48 hpi, respectively. Among 24 and 56 significantly enriched pathways, 4 and 13 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. More immune-related signaling pathways including Leukocyte transendothelial migration, Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, TNF signaling pathway, Complement and coagulation cascades, Hematopoietic cell lineage and Jak-STAT signaling pathway were differently enriched for upregulated DEGs at 48 hpi, which were completely different from that in the intestines. Furthmore, comparative transcriptome analysis revealed that the downregulated 1618 genes and upregulated 1622 genes existed both at 24 hpi and 48 hpi for the intestine samples. In the skins, the downregulated 672 genes and upregulated 428 genes existed both at 24 hpi and 48 hpi. Three pathways related to immune processes were significantly enriched for downregulated DEGs both in the intestines and skins collected at 24 hpi and 48 hpi, which included Antigen processing and presentation, Intestinal immune network for IgA production and Hematopoietic cell lineage. Interaction network analysis of DEGs identified the main DEGs in the sub-network of complement and coagulation cascades both in the intestines and skins. Twenty of DEGs involved in complement and coagulation cascades were further validated by Real-time quantitative PCR. Altogether, the results obtained in this study will provide insight into the immune response of zebrafish against S. agalactiae XQ-1 infection in fatal conditions, and reveal the discrepant expression pattern of complement and coagulation cascades in the intestines and skins.

RIP2 Is a Critical Regulator for NLRs Signaling and MHC Antigen Presentation but Not for MAPK and PI3K/Akt Pathways.

RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as "Antigen processing and presentation" and "NOD-like receptor signaling pathway" and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.

NOD1 deficiency impairs CD44a/Lck as well as PI3K/Akt pathway.

Pattern recognition receptors (PRRs) are crucial for host defense and tissue homeostasis against infecting pathogens. PRRs are highly conserved cross species, suggesting their key roles in fundamental biological processes. Though much have been learned for NOD1 receptor in the innate and adaptive immune responses, the roles of NOD1 during embryonic and larval stages remain poorly understood. Here, we report that NOD1 is necessary for the modulation of PI3K-Akt pathway and larval survival in zebrafish. Transcriptome analysis revealed that the significantly enriched pathways in NOD1 -/- zebrafish larvae were mainly involved in metabolism and immune system processes. Biochemical analysis demonstrated that NOD1 was required for the expression of CD44a that, in turn, activated the PI3K-Akt pathway during larval development. Conversely, over-expression of CD44a in NOD1-deficient zebrafish restored the modulation of the PI3K-Akt pathway and improved larval survival. Collectively, our work indicates that NOD1 plays a previously undetected protective role in larval survival through CD44a-mediated activation of the PI3K-Akt signaling.

Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

[The role of Src kinase inhibitor ZD6474 on multi-drug resistant K562/A02 cells].

OBJECTIVE: To investigate the role of Src kinase inhibitor ZD6474 on the growth of multidrug-resistant K562/A02 cells and its regulatory mechanisms. METHODS: The possible mechanisms of drug-resistance were tested by Western blot. Proliferation assays and cell cycle distribution were analyzed by WST metric analysis. Western blot were used to investigate the mechanisms of antiproliferative activity induced by tyrosine kinase inhibitor ZD6474. The in vivo anti-tumor activity was evaluated in K562, K562/A02 xenografted nude mice by administration of ZD6474 (25 - 100 mg×kg(-1)×d(-1), PO). RESULTS: Compared with parental K562 cells, marked high levels of p-Src and Src expression were detected in K562/A02 cells. WST results showed that the IC(50) values of ZD6474 on K562 and K562/A02 after 48 hours incubation were (1.61 ± 0.07) µmol/L and (3.22 ± 0.21)µmol/L, respectively. ZD6474 caused an accumulation of cells in the G(0)/G(1) fraction and apoptosis by inhibiting the expressions of p-Src and Src kinase. Administration of ZD6474 produced a dose-dependent inhibition of tumor growth. 50 mg/kg ZD6474 produced the growth inhibition rates of 43.7% and 56.3%, respectively in K562 and K562/A02. CONCLUSION: Our results indicated that inhibiting Src kinase could induce K562/A02 cells apoptosis in vitro and in vivo.

Fingerprint analysis of placenta polypeptide injection by high performance liquid chromatography.

Objective: To develop the representative fingerprint for the quality control of placenta polypeptide injection. METHODS: The chromatographic separation was performed using a Phenomenex Gemini C18 column (250 mm×4.6 mm, 5 µm) maintained at 30 °C. 0.1% aqueous trifluoroacetic acid (Solvent A) and acetonitrile contained 0.1% TFA (Solvent B) were used as mobile phase with a gradient elution. Detection wavelength was 280 nm with the sample injection volume of 50 µL; the flow rate was 1.0 mL/min. The fingerprints of different samples were investigated by similarity analysis. RESULTS: Nine peaks were identified as the characteristic common peaks. The similarities of the fingerprints of the 10 batches of samples were above 0.992. CONCLUSION: This method showed high precision and good repeatability, and provided the basis for the improvement of the quality control of placenta polypeptide injection.

[Repair of finger pulp defect with the homodigital spiral neurovascular island flap].

OBJECTIVE: To investigate the method and clinical outcome of reconstructing large pulp defects of the fingertips with a homodigital neurovascular island flap. METHODS: Form June 2007 to October 2009, 16 patients with the defects of pulp were repaired by the spiral flap that was a homodigital neurovascular island flap with a unique spiral advancement and transposition design allowed pulp reconstruction using sensate glabrous skin while restricting donor morbidity to the injured digit. There were 12 males and 4 females with an average age of 37 years ranging from 18 to 49 years. The defect were caused by machine crush injury 11 cases, pressure injury by heavy objects in 2 cases, crush injury by door of car 1 case, injury by saw in 2 cases. The defect was located in the index finger in 5 cases, the middle finger in 9 cases,the ring finger in 2 cases. All injuries had large pulp defects averaging 1.6 cm (long) x 1.2 cm (wide) to 2.5 cm (long) x 1.7 cm (wide). Short-term results for all patients were reviewed. Outcome measures included static 2-point discrimination, total active motion, and hypersensitivity or cold intolerance. RESULTS: All flaps achieved primary healing with no complications. Sensory recovery was excellent with an average 2-point discrimination of 5.1 mm. All patients were followed-up for 8 to 20 months (averaged 12 months) with highly satisfactory with both aesthetic and functional outcome. There was no hypersensitivity or cold intolerance. According to the evaluation of total active motion (TAM) scales, the results were excellent in 7 cases, good in 8 cases (9 lesions) and fair in 1 case. CONCLUSION: The spiral advancement-transposition flap is suitable for resurfacing large pulp defects with excellent short-term functional and aesthetic results and high patient satisfaction.

[Effect of ZD6474 on the proliferation of imatinib-resistant K562 cells].

OBJECTIVE: To investigate the effect of tyrosine kinase inhibitor ZD6474 (Vandetanib) on the proliferative inhibition of K562 cells and its derived imatinib-resistant K562/G cells and its mechanism. METHODS: Imatinib-resistant K562/G cells were obtained by culturing cells in gradually increasing concentrations of imatinib. The changed factors related to drug-resistance were tested by Western blot. ZD6474 and imatinib affected K562/G and parental K562 cells proliferation were analyzed by WST assay. Flow cytometry was used to analyze cell cycle. Direct inhibition of Src activity by ZD6474 was measured by a colorimetric ELISA assay with recombinant human Src kinase. RESULTS: 10 µmol/L imatinib failed to inhibit K562/G cells proliferation or induce cell cycle arrest. Compared with that in parental K562 cells, there were marked high levels of p-Src and Src protein in K562/G cells. The expression of Bcl-2 and p-STAT3 also increased in K562/G cells. After 48 hours incubation, the IC(50) values of ZD6474 in K562 and K562/G cells were 1.61 µmol/L and 3.18 µmol/L, respectively. ZD6474 treatment caused accumulation of cells in the G(0)/G(1) fraction and cell apoptosis in K562 and K562/G cells. ZD6474 decreased the expression of p-Src and Src at post-transcriptional level. Moreover, ZD6474 increased the ratio of Bax/Bcl-2 and decreased the expression of p-STAT3 at the same concentration for inducing apoptosis. CONCLUSIONS: ZD6474 is effective in inhibiting the proliferation of imatinib-resistant K562/G cells and parental K562 cells, and induces their apoptasis by significant inhibition of Src kinase activity. Our study provides a reliable experimental basis for chronic myeloid leukemia treatment with ZD6474.